(1) Field of the Invention
The present invention relates to a peptide mimotope of the non-peptide mycotoxin deoxynivalenol. In particular, the peptide mimotope competes with deoxynivalenol for binding to a monoclonal antibody, is antagonistic to the inhibitory effects of deoxynivalenol on in vitro protein synthesis, and does not elicit antibodies in mice that recognize the deoxynivalenol. The present invention also relates to a method that uses the peptide mimotope to determine whether corn, grains or mixed feed is contaminated with fungi that produces deoxynivalenol. The present invention further relates to transgenic plants resistant to deoxynivalenol.
(2) Description of Related Art
Deoxynivalenol (DON) or vomitoxin or dehydronivalenol is 12,13-epoxy-3,7,15-trichothec-9-en-8-one, which is a mycotoxin of the 12,13-epoxy-trichothecenes class of sesquiterpene mycotoxins. It is produced primarily by the fungus Gibberella zeae (Schwein.) Petch (anamorph=Fusarium graminearum Schwabe), which infects corn, small grains and mixed feeds (Hart et al., J. Agric. Food Chem. 31: 657-659 (183); Hart et al., Plant Dis. 66: 1133-1135 (1982); Neish et al., Can. J. Plant Sci. 61: 811-815 (1981)). At the cellular level, the primary toxic effect of DON is inhibition of protein synthesis by binding to the 60S ribosomal subunit, which interferes with peptidyltransferase (Betina, Chem. Biol. Interact. 71: 105-146 (1989); Weber et al., Biochem. 31: 9350-9354 (1992)). DON can cause anorexia and emesis in animals (Scott et al. Proc. natl. Acad. Sci. USA 89: 5398-5402 (1992)). Other toxic effects of DON include skin irritation, hemorrhaging, hematological changes, human lymphocyte blastogenesis impairment, radiomimetic effects, apoptosis and immunotoxicity (Scott et al. ibid.).
DON is primarily found as a contaminant in grains that are infected with the above fungi. It has also been implicated as a chemical warfare agent. Currently, the only means for eliminating DON from human and animal foodstuffs is to detect DON in food and to remove any contaminated foodstuffs from the food supply. Immunoassays offer several advantages compared to other analytical methods for detecting DON in foodstuffs. Following the development of the first monoclonal antibody to DON (Casale et al., J. Agric. Food Chem. 36: 663-668 (1988)), immunological methods, primarily enzyme-linked immunosorbant assay (ELISA), have been widely used for detection of DON (Pestka et al., Food Technol. 49: 120-128 (1995)). An immunoassay for trichothecenes such as DON is disclosed in U.S. Pat. No. 4,879,248 to Chu et al. and kit comprising the immunoassay is disclosed in U.S. Pat. No. 5,118,612 to Chu et al. The immunoassay and kit are either radio immunoassays (RIA) or enzyme-linked immunosorbant assay ELISA based on a competitive control that is DON. These immunological assays have advantages which include high specificity, ease of use, facile sample preparation, and good sensitivity.
The disadvantages of these immunoassays is that they require the user to handle purified DON which poses a toxicity risk to the user. In addition, chemical conjugation of DON to a carrier protein or an enzyme has low efficiency because it involves extensive modification and blocking stages and causes substantial bridge-group interferences and un-wanted cross-reactions (Casale et al., ibid.: Pestka et al., ibid.; Yuan et al., Appl. Environ. Microbiol. 63: 263-269 (1997)). Furthermore, DON is poorly immunogenic and when DON is conjugated to a carrier protein, it's immunogenicity is only marginally enhanced.
Therefore, it is desirable that an alternative to DON be developed. Preferably, the DON alternative would be non-toxic to the user, not require conjugation to a protein, and be highly immunogenic.